PROJECT SUMMARY/ABSTRACT The hematopoietic system is the foundation of the inflammatory response. Hematopoietic stem cells and multipotent progenitors (collectively termed HSPCs) residing in adult bone marrow generate inflammatory myeloid and lymphoid populations. HSPCs also respond to pathogen-associated inflammatory stimuli by adjusting the output of cells required to fight infection. HSPC activity is unfortunately co-opted in chronic inflammatory conditions. These induce HSPC deregulation, loss of long-term stem cell repopulating function, DNA damage, and myeloid-skewed hematopoiesis. In humans, chronic inflammation is linked with bone marrow failure, myelodysplastic syndrome, and acute myeloid leukemia. An important gap in knowledge is how the hematopoietic system is protected from the harmful effects of inflammation. By investigating this question, we discovered a key role for the cytokine-activated transcriptional regulator STAT3; specifically, our results suggest STAT3 anti-inflammatory activity is necessary to preserve HSPCs and lineage-balanced hematopoiesis. We showed previously that STAT3 anti-inflammatory activity is mediated by transcriptional repression of Ube2n, encoding Ubc13, an E2 ubiquitin-conjugating enzyme that activates TRAF6 and NF- ?B/MAPK signaling downstream of Toll-like receptors (TLRs) (e.g., TLR2, TLR4). We found hematopoietic- restricted Stat3-deficiency leads to bone marrow failure, HSPC loss, myeloid-skewed hematopoiesis and increased progenitor pro-inflammatory signaling. Strikingly, concomitant removal of Ube2n from Stat3-deficient hematopoietic cells enables hematopoietic activity, suggesting STAT3 restraint of Ubc13 is required to maintain functional HSPCs. Thus, we hypothesize STAT3 has a central role in protecting HSPCs from inflammation-induced damage via modulation of Ubc13. To test this (Aim 1), we will investigate the role for STAT3 in protecting hematopoietic function in inflammation. Using mixed bone marrow chimeras, we will determine roles for STAT3 and Ubc13 in hematopoietic responses to inflammation; we will investigate STAT3- activating cytokines and producer populations in bone marrow; and we will test the requirement for STAT3 transcriptional activity in HSPCs during inflammation using a transgenic strain expressing a transcriptionally defective STAT3 mutant. In Aim 2, we will delineate molecular pathways by which STAT3 protects HSPCs from inflammation-induced damage. We will determine roles for STAT3 and Ubc13 in protecting HSPCs from DNA damage and loss of quiescence during inflammation, and we will determine STAT3- and Ubc13- genome- wide transcriptional responses in HSPCs in homeostasis and inflammation. We anticipate this project will provide unprecedented insight into intrinsic HSPCs protective mechanisms, fundamental information that will move the field forward to an improved understanding of immune system regulation during inflammation.